No description available.

No description available.

All Strychnos and Aspidosperma alkaloids possess a core pyrrolo[2,3-d]carbazole ABCE tetracycle. In order to develop an efficient and divergent methodology for the synthesis of Strychnos alkaloids, a streamlined synthetic sequence to the ABCE tetracycle has been developed. It features a Mitsunobu activation of an N-hydroxyethyl gramine intermediate and subsequent intramolecular aza-Baylis-Hillman reaction. This method was first applied in the total synthesis of (±)-alstolucine B. Additional key steps in the synthesis included (1) chemoselective intermolecular and intramolecular Michael additions and (2) a Swern indoline oxidation. The second application of this method was in the first total synthesis of (-)-melotenine A, a novel rearranged Aspidosperma alkaloid with potent biological activity. Additional key steps in the synthesis included (1) a Piers annulation of a vinyl iodide and a methyl ketone to prepare the D ring and (2) a site-selective intermolecular vinylogous aldol reaction / Chemistry

Tall fescue [Schedonorus arundinaceus (Schreb.) Dumort.] is an important forage crop in the United States and covers over 14 million ha. The presence of Neotyphodium coenophialum, an endophytic fungus in tall fescue, is associated with several disorders in grazing livestock, but also increased persistence of tall fescue. These disorders, commonly called fescue toxicosis, are responsible for large economic losses in the beef cattle industry each year. This research examined the effect of three fescue types [endophyte-infected Kentucky 31 tall fescue (E+), endophyte-free Kentucky 31 tall fescue (E-), non-ergot alkaloid-producing endophyte Q4508-AR542 tall fescue (Q)], and Lakota prairie grass (L; Bromus catharticus Vahl.) on animal response, alkaloid appearance, DMI, and copper status. Ergovaline (EV) is the most abundant ergot alkaloid in tall fescue and has previously been considered the causative toxin in fescue toxicosis. More recently it is simpler ergot alkaloids, such as lysergic acid amide (LSA) have been implicated. The objective of the first project was to evaluate animal performance and alkaloid (EV and LSA) appearance in forage and ruminal fluid of steers grazing E-, Q, E+, and L. Average daily gains were greater (P < 0.05) on E-, Q and L compared to E+, and there was a trend (P = 0.11) for gains on E- to be higher than with Q. The seasonal appearance of LSA in ruminal fluid was similar to the seasonal pattern of alkaloids in E+ forage. Ergovaline was not detectable in ruminal fluid of steers grazing E+. Alkaloids were not detectable in forage or ruminal fluid of steers grazing E-, Q, or L. The appearance of LSA in ruminal fluid of steers grazing E+ suggests that this alkaloid may contribute to fescue toxicosis. Low DMI of animals grazing E+ tall fescue is considered a key factor in decreased animal performance compared to other fescue types. The objective of the second project was to evaluate DMI of steers grazing E-, E+, Q, and L pastures using the alkane technique. Dry matter intake of steers grazing E- was greater (P < 0.001) than Q, E+, and L and DMI of steers grazing Q and E+ were similar (P > 0.10) in 2004. In 2005, DMI did not differ (P = 0.23) among fescue types. These results suggest that decreased DMI effects ADG of steers grazing E+ compared to those grazing E-, and lower DMI of Q suggests that the fescue variety Q4508 may not be the optimal variety for the incorporation on non-ergot alkaloid-producing endophytes. Reactive oxygen metabolites such as superoxide (O₂⁻) are produced by both endogenous and exogenous sources and an accumulation of these compounds can result in oxidative stress. Copper/zinc superoxide dismutase (SOD) is a Cu-based antioxidant metalloprotein that acts as a defense against oxidative stress by the scavenging of O₂⁻. <i>Neotyphodium</i>-infected tall fescue is typically lower in Cu which could potentially increase oxidative stress of animals grazing this forage. Therefore the objective of the third project was to investigate the Cu and SOD status of steers grazing E-, E+, Q, and L forages. Copper levels of all forages were below the dietary requirement (10μg Cu/g DM) of growing cattle. In 2004, steers grazing E+ exhibited lower (P <0.05) liver Cu compared to E- and Cu intake was lower (P < 0.001). Cu/Zn SOD enzymatic activity and mRNA relative expression did not differ (P > 0.10) among treatments. Copper intake of steers grazing E+ tall fescue was sufficient to maintain, but not replenish liver Cu, and SOD status did not appear compromised by grazing E+ at these Cu levels. / Ph. D.

A study of the oxidation reactions of annotinine lactamdiol provided chemical evidence that ring A of annotinine was sixmembered and confirmed that the ether ring in annotinine was an epoxide ring. The detailed structure of ring B of annotinine was elucidated through a study of the reactions of diphenyldesoxodihydroannotinine. Unsuccessful attempts were made to correlate the structure of annotinine with the structures of lycopodine and other Lycopodium alkaloids. / Dissertation / Doctor of Philosophy (PhD)

A general methodology for the synthesis of pyrrolizidine alkaloids has been developed. The two key steps in the sequence were the synthesis of the dienic azide system 1 and its regioisomer 2 by utilizing the vinylogous Reformatsky reaction, and the intramolecular additions of the azide moiety across the activated dienes with subsequent pyrolyses to provide the pyrrolizidines of type 3. This study broadened the scope of the vinylogous Reformatsky reaction to include the preparation of alkaloid synthons and introduced the heteroatom equivalent of the carbenoid [4+1] annulation as a method or alkaloid synthesis. [See docment for associated image] / M.S.

Gold catalysis has attracted much attention within the chemical community in recent years, and its importance as a synthetic tool has only started to be uncovered. This thesis describes the development of a gold(I) catalyzed transformation and its application to the synthesis of a structurally unique Lycopodium alkaloid, Magellanine. Although there have been a few reports on the synthesis of the magellanane core to date, the approach described herein would represent a new and efficient strategy to construct the angularly fused tetracyclic core. The 5 exo dig/Prins reaction that would be the key step of the synthesis was first developed and studied on a model substrate, enabling the verification of the hypothesis that this transformation could indeed form the A and B rings of Magellanine and be applied to its synthesis. This reaction formed the tricyclic products in good yields and in good exo:endo ratios. The synthesis of Magellanine was undertaken, but problems of isomerization prevented the synthesis of the desired 5 exo dig/Prins substrate, which contained the C and D rings of Magellanine with a cis relationship at the ring junction. However, an almost identical substrate, save for a trans configuration between the C and D rings instead of the cis configuration, was prepared and served in further establishing the applicability of this methodology to the synthesis of Magellanine by successfully undergoing the 5-exo-dig/Prins reaction and generating the tetracyclic products. Studies of the steps following the key transformation were performed on the model substrate, allowing for the evaluation of these steps prior to their use in the synthesis. The results of the studies indicate a possible need to revisit the order in which the steps should be carried out. Promising solutions to the different obstacle encountered during the work are presented, demonstrating how the synthesis of Magellanine through a route featuring the 5-exo-dig/Prins cyclization is attainable.

神經母細胞瘤是一種交感神經系統的腫瘤。它是最常見的兒童顱外實體瘤。神經母細胞瘤約佔兒童腫瘤的8-10%，佔15%的兒童腫瘤死亡率。目前神經母細胞瘤的治療方法包括外科手術， 化學藥物治療， 放射治療， 幹細胞移植， 誘導分化治療和免疫治療。 然而，這些治療方法通常會導致許多無法避免的嚴重的副作用。因此，開發能高效抑制神經母細胞瘤但對正常細胞無明顯副作用的新型藥物顯得至關重要。最近，用來源於天然產物或中藥的化合物治療癌症引起了科學家的廣泛關注。靛玉紅-3’-肟（Indirubin-3’-oxime, I3M）和色胺酮（tryptanthrin）分別是從板藍根中分離得到的靛藍生物鹼和吲哚喹唑啉類生物鹼。據研究報導，這兩種生物鹼具有多種生物學和藥理學作用，包括抗菌，抗炎症和抗腫瘤作用。它們對體外的多種人腫瘤細胞具有抗腫瘤作用。然而，它們對人神經母細胞瘤的調節作用和作用機理仍不太清楚。在我的博士研究課題中，我們對板藍根生物鹼包括靛玉紅-3’-肟和色胺酮對人神經母細胞瘤的抗腫瘤活性和作用機制進行了研究和探討。 / 首先，我們研究了靛玉紅-3’-肟對人神經母細胞瘤的抗腫瘤活性和作用機制。實驗結果表明，靛玉紅-3’-肟能夠抑制人神經母細胞瘤LA-N-1, SH-SY5Y 和 SK-N-DZ細胞系的生長，並且其抑制效果呈時間和濃度依賴性。然而，靛玉紅-3’-肟對正常細胞無顯著的細胞毒性作用。對其生長抑制作用機制的研究結果表明靛玉紅-3’-肟能夠特異性地減少LA-N-1細胞系中線粒體的調節子ERR和 PGC-1的表達，從而導致線粒體生成減少，線粒體膜電位降低以及線粒體活性氧（ROS）增加。靛玉紅-3’-肟還增加週期蛋白依賴性蛋白激酶（CDK）抑制蛋白p27{U+1D37}{U+2071}{U+1D56}¹的蛋白水平並降低週期蛋白依賴性蛋白激酶2（CDK2）和細胞週期蛋白E（Cyclin E）的表達，從而導致細胞週期阻滯在G0/G1期。 另外，我們發現靛玉紅-3’-肟能減少SH-SY5Y細胞系的線粒體生成，增加細胞內活性氧的水準從而導致細胞週期停滯在G0/G1期和細胞凋亡。以上結果表明靛玉紅-3’-肟可能通過破壞線粒體的功能從而導致LA-N-1和SH-SY5Y細胞的細胞週期阻滯和誘導SH-SY5Y細胞的細胞凋亡來發揮其抗腫瘤的作用。 / 接著，我們對色胺酮對人神經母細胞瘤的抗腫瘤活性和作用機制進行了探討。我們研究的結果表明，色胺酮可以時間和濃度依賴性地抑制人神經母細胞瘤LA-N-1, SH-SY5Y 和 SK-N-DZ細胞系的生長，而對正常的細胞無顯著的細胞毒性。對色胺酮抑制人神經母細胞瘤生長的機制研究表明，色胺酮能顯著地降低細胞週期蛋白（Cyclin D1和 Cyclin D3）和週期蛋白依賴性蛋白激酶（CDK4和CDK6）的蛋白水平從而導致細胞週期停滯在G0/G1期。色胺酮可以激活半胱氨酸天冬氨酸蛋白酶8,9和3/7（caspase 8, caspase 9 和 caspase 3/7）從而誘導LA-N-1細胞凋亡。色胺酮還可以誘導LA-N-1細胞分化，表現為神經細胞分化的細胞形態，乙醯膽鹼酯酶活性的增加和多種細胞分化的分子標記的表達上調。另外，色胺酮還能降低LA-N-1細胞中N-myc的表達。有趣的是，通過RNA干擾技術降低N-myc的表達能誘導LA-N-1細胞的分化。總的來說，以上結果顯示色胺酮通過誘導細胞週期阻滯，細胞凋亡和細胞分化從而發揮其抗腫瘤的作用。它可能被開發為治療有N-myc基因擴增的高危的人神經母細胞瘤的潛在藥物。 / 此外，我們還研究了靛玉紅-3’-肟和色胺酮是否具有抗血管生成的作用。體外實驗的研究結果表明，靛玉紅-3’-肟和色胺酮能夠濃度依賴性地抑制人微血管上皮細胞 （HMEC-1細胞）的增殖，遷徙和血管生成，但對HMEC-1細胞卻沒有顯著的細胞毒性作用。此外，靛玉紅-3’-肟和色胺酮能顯著地抑制小鼠體內的基質膠栓（Matrigel plug）的血管生成。對它們抑制血管生成的機制的研究表明，靛玉紅-3’-肟能下調血管生成素1（Ang-1）和基質金屬蛋白酶2（MMP2）的表達，上調血管生成素2（Ang-2）的表達。靛玉紅-3’-肟能結合到血管內皮生長因數受體2（VEGFR2） 的ATP結合位點上從而抑制血管內皮生長因數受體2的磷酸化和下游的MEK/ERK和PI3K/AKT/GSK信號轉導通路。色胺酮同樣可以抑制多種血管生成因子（Ang-1,PDGFB 和MMP2）的表達。此外，它可以結合到血管內皮生長因數受體2 的ATP結合位點上從而抑制血管內皮生長因數受體2的磷酸化和血管內皮生長因數受體2介導的ERK1/2信號通路。以上的體外和體內實驗研究結果表明靛玉紅-3’-肟和色胺酮通過靶向血管內皮生長因數受體2介導的信號通路來發揮其抗血管生成的作用。它們可能被開發為治療血管生成相關疾病的潛在藥物。 / 總而言之，我們的研究結果表明靛玉紅-3’-肟和色胺酮通過誘導人神經母細胞瘤細胞的細胞週期阻滯，細胞凋亡或誘導神經細胞分化從而抑制人神經母細胞瘤細胞的生長。然而，它們對正常細胞無顯著的細胞毒性作用。此外，靛玉紅-3’-肟和色胺酮通過靶向血管內皮生長因數受體2介導的信號通路來發揮其抗血管生成的作用。未來的研究將進一步探討靛玉紅-3’-肟和色胺酮對人神經母細胞瘤細胞的分子作用機理。另外，通過人神經母細胞瘤細胞的裸鼠移植瘤動物模型可進一步去了解這些板藍根生物鹼在體內的抗腫瘤效果。 / Neuroblastoma, a tumor of the sympathetic nervous system, is the most common extracranial solid cancer in childhood. It accounts for 8% to 10% of all childhood cancers and for approximately 15% of cancer deaths in children. Current treatment modalities consist of surgery, chemotherapy, radiation therapy, stem cell transplantation, differentiation therapy and immunotherapy. However, these treatments often cause severe and inevitable side effects. It is important to develop novel drugs with higher efficacy on neuroblastoma cells and minimal side effects on normal cells. The use of new promising therapeutic compounds derived from natural products or Chinese herbs have attracted much attention of scientist as an alternative strategy in cancer treatment. Indirubin-3’-oxime (I3M) is an indigo alkaloid and tryptanthrin is an indoloquinazoline alkaloid which can be isolated from the dried roots of medicinal indigo plants known as Banlangen. These two alkaloids have been reported to possess various biological and pharmacological activities, such as anti-microbial, anti-inflammatory, and anti-tumor effects. They were found to exhibit potent anti-tumor activities on various types of human cancer cells in vitro. However, their modulatory effects on human neuroblastoma and the underlying mechanisms remain poorly understood. In my PhD project, the possible anti-tumor activities and action mechanisms of Banlangen alkaloids, including I3M and tryptanthrin, on human neuroblastoma cells were investigated. / Firstly, the anti-cancer effects of I3M on human neuroblastoma cells and the underlying mechanisms were investigated. I3M was found to inhibit the growth of the human neuroblastoma LA-N-1, SH-SY5Y and SK-N-DZ cells in a concentration- and time-dependent manner, but exhibited little, if any, direct cytotoxicity on normal cells. Mechanistic studies showed that I3M specifically decreased the expression of mitochondrial regulators ERRγ and PGC-1βand resulted in decreased mitochondrial mass and altered mitochondrial function characterized by reduction in mitochondrial membrane potential and elevation of reactive oxygen species (ROS) level in LA-N-1 cells. I3M also increased the level of cyclin-dependent kinase (CDK) inhibitor p27{U+1D37}{U+2071}{U+1D56}¹ and reduced the levels of CDK2 and cyclin E in LA-N-1 cells, leading to cell cycle arrest at the G0/G1 phase. In addition, I3M was also found to reduce the mitochondrial mass and increase the ROS level leading to cell cycle arrest at G0/G1 phase and apoptosis in SH-SY5Y cells. These results, when taken together, suggest that I3M might exert its anti-tumor activity by causing mitochondrial dysfunction which led to cell cycle arrest in LA-N-1 cells and resulted in cycle arrest and apoptosis in SH-SY5Y cells. / The anti-tumor effects and action mechanisms of tryptanthrin on the human neuroblastoma cells were also examined. Our results showed that tryptanthrin inhibited the growth of the human neuroblastoma LA-N-1, SH-SY5Y and SK-N-DZ cells in a concentration- and time-dependent manner, but exhibited little, if any, direct cytotoxicity on normal cells. Mechanistic studies indicated that tryptanthrin significantly reduced the protein levels of cyclin D1, cyclin D3, CDK4 and CDK6 leading to cell cycle arrest at G0/G1 phase. In addition, tryptanthrin activated caspase 8, caspase 9 and caspase 3/7 resulting in apoptosis of the human neuroblastoma LA-N-1 cells. Moreover, tryptanthrin induced neuronal differentiation of LA-N-1 cells, as assessed by morphological criteria, enhancement of acetylcholine esterase activity and up-regulation of various differentiation markers. Tryptanthrin treatment also led to the significant reduction of N-myc expression in LA-N-1 cells. Interestingly, down-regulating N-myc expression using siRNA induced neuronal differentiation of LA-N-1 cells. Collectively, these results indicate that tryptanthrin might exert its anti-tumor activity on the human neuroblastoma LA-N-1 cells by inducing cell cycle arrest, apoptosis and neuronal differentiation. It might be exploited as a potential therapeutic candidate for the treatment of high-risk neuroblastomas with N-myc-amplification. / Moreover, the anti-angiogenic activities of I3M and tryptanthrin were studied. Our results showed that I3M and tryptanthrin inhibited the proliferation, migration, and tube formation of the human microvascular endothelial HMEC-1 cells in vitro in a concentration-dependent manner but exhibited no significant cytotoxicity on these cells. Moreover, I3M and tryptanthrin markedly suppressed the in vivo angiogenesis in Matrigel plugs in mice. Mechanistic studies indicated that I3M down-regulated the expression of Ang-1 and MMP2 and up-regulated the expression of Ang-2. It also bound to the ATP-binding site of VEGFR2 and inhibited the phosphorylation of VEGFR2 leading to suppression of the down-stream MEK/ERK and PI3K/AKT/GSK signaling pathways in HMEC-1 cells. Similarly, tryptanthrin also reduced the expression of several angiogenic factors (Ang-1, PDGFB and MMP2) in HMEC-1 cells. In addition, tryptanthrin also bound to the ATP-binding site of VEGFR2 and suppressed the phosphorylation of VEGFR2 and VEGFR2-mediated ERK1/2 signaling pathway in HMEC-1 cells. Collectively, our results demonstrated that I3M and tryptanthrin exhibited anti-angiogenic activity both in vitro and in vivo by specifically targeting the VEGFR2-mediated signaling pathways and might be exploited as potential therapeutic candidates for the treatment of angiogenesis-related diseases. / In conclusion, our findings indicate that I3M and tryptanthrin might exert their growth-inhibitory effect on the human neuroblastoma cells by causing cell cycle arrest, inducing apoptosis or inducing neuronal differentiation. However, they exhibited minimal cytotoxicity towards the normal cells. Moreover, I3M and tryptanthrin were found to possess anti-angiogenic activities by targeting the VEGFR2-mediated signaling pathways. In the future, investigations should be focused on further elucidation of the molecular action mechanisms of I3M and tryptanthrin on human neuroblastoma cells and to test the anti-tumor efficacy of I3M and tryptanthrin in animal models, using human neuroblastoma xenografts in nude mice. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liao, Xuemei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 206-229). / Abstract also in Chinese. / Acknowledgments --- p.i / Abbreviations --- p.ii / Publications --- p.vi / Abstract --- p.vii / 摘要 --- p.xii / Table of Contents --- p.xvi / Chapter One / General Introduction --- p.1 / Chapter 1.1 --- Neuroblastoma --- p.2 / Chapter 1.1.1 --- Epidemiology of neuroblastoma --- p.2 / Chapter 1.1.2 --- Classification of neuroblastoma --- p.6 / Chapter 1.1.3 --- Clinical symptoms and diagnosis of neuroblastoma --- p.10 / Chapter 1.1.4 --- Molecular pathogenesis of neuroblastoma --- p.13 / Chapter 1.1.4.1 --- Genetic alterations in neuroblastoma --- p.13 / Chapter 1.1.4.2 --- Disruption of cell division cycle, apoptotic and signaling pathways --- p.16 / Chapter 1.1.5 --- Treatment strategies --- p.19 / Chapter 1.1.5.1 --- Low-risk neuroblastoma treatment strategy --- p.19 / Chapter 1.1.5.2 --- Intermediate-risk neuroblastoma treatment strategy --- p.20 / Chapter 1.1.5.3 --- High-risk neuroblastoma treatment strategy --- p.21 / Chapter 1.1.5.4 --- Side effects of treatment --- p.23 / Chapter 1.2 --- Banlangen alkaloids --- p.23 / Chapter 1.2.1 --- Overview of Banlangen alkaloids --- p.23 / Chapter 1.2.2 --- Biological and pharmacological effects of Banlangen alkaloids --- p.28 / Chapter 1.2.2.1 --- Anti-inflammatory activity --- p.28 / Chapter 1.2.2.2 --- Anti-microbial activity --- p.29 / Chapter 1.2.2.3 --- Anti-tumor activity --- p.30 / Chapter 1.2.2.4 --- Other biological activities --- p.32 / Chapter 1.2.3 --- Bioavailability of Banlangen alkaloids --- p.33 / Chapter 1.2.4 --- Toxicity of Banlangen alkaloids --- p.34 / Chapter 1.3 --- Aims and scope of this project --- p.36 / Chapter Two / Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Animals --- p.39 / Chapter 2.1.2 --- Cell lines --- p.39 / Chapter 2.1.3 --- Cell culture media --- p.41 / Chapter 2.1.4 --- Drugs and chemicals --- p.42 / Chapter 2.1.5 --- Reagents and buffers for cell culture --- p.44 / Chapter 2.1.6 --- General staining solutions --- p.47 / Chapter 2.1.7 --- Reagents and buffers for cell growth assays --- p.48 / Chapter 2.1.8 --- Reagents and buffers for flow cytometry --- p.48 / Chapter 2.1.9 --- Reagents and buffers for acetylcholine esterase activity assay --- p.50 / Chapter 2.1.10 --- Reagents and buffers for immunocytochemistry --- p.51 / Chapter 2.1.11 --- Reagents and buffers for total RNA extraction --- p.53 / Chapter 2.1.12 --- Reagents and buffers for reverse transcription --- p.54 / Chapter 2.1.13 --- Reagents for quantitative real-time polymerase chain reaction (qRT-PCR) --- p.56 / Chapter 2.1.14 --- Reagents and buffers for Western blotting --- p.59 / Chapter 2.1.15 --- Assay kits --- p.65 / Chapter 2.2 --- Methods --- p.68 / Chapter 2.2.1 --- Culture of cells --- p.68 / Chapter 2.2.2 --- MTT assay --- p.69 / Chapter 2.2.3 --- Cell proliferation assay --- p.70 / Chapter 2.2.4 --- Trypan blue exclusion test --- p.70 / Chapter 2.2.5 --- Cytotoxicity assay --- p.71 / Chapter 2.2.6 --- Colony-forming assay --- p.72 / Chapter 2.2.7 --- Cell cycle analysis --- p.72 / Chapter 2.2.8 --- Assessment of apoptosis --- p.73 / Chapter 2.2.9 --- Caspase activity determination --- p.74 / Chapter 2.2.10 --- Mitochondrial mass assay --- p.75 / Chapter 2.2.11 --- Reactive oxygen species (ROS) assay --- p.75 / Chapter 2.2.12 --- Mitochondrial membrane potential determination --- p.76 / Chapter 2.2.13 --- Morphological detection of cell differentiation --- p.76 / Chapter 2.2.14 --- Acetylcholine esterase activity determination --- p.77 / Chapter 2.2.15 --- Immunocytochemistry --- p.77 / Chapter 2.2.16 --- RNA interference --- p.78 / Chapter 2.2.17 --- Wound healing assay --- p.79 / Chapter 2.2.18 --- Tube formation assay --- p.79 / Chapter 2.2.19 --- In vivo Matrigel plug assay --- p.80 / Chapter 2.2.20 --- Phospho-VEGFR2 Sandwich ELISA assay --- p.80 / Chapter 2.2.21 --- Isolation of total cellular RNA --- p.81 / Chapter 2.2.22 --- Reverse transcription (RT) --- p.82 / Chapter 2.2.23 --- Quantitative real-time PCR --- p.83 / Chapter 2.2.24 --- Total protein extraction --- p.84 / Chapter 2.2.25 --- Protein concentration determination --- p.84 / Chapter 2.2.26 --- Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) --- p.85 / Chapter 2.2.27 --- Semi-dry Western blotting --- p.85 / Chapter 2.2.28 --- Enhanced chemiluminescence (ECL) assay --- p.87 / Chapter 2.2.29 --- Molecular docking --- p.87 / Chapter 2.2.30 --- Statistical analysis --- p.88 / Chapter Three / Modulatory effects and action mechanisms of indirubin-3'-oxime on human neuroblastoma cells --- p.89 / Chapter 3.1 --- Introduction --- p.90 / Chapter 3.2 --- Results --- p.94 / Chapter 3.2.1 --- Indirubin-3’-oxime inhibited the growth and colony formation of human neuroblastoma cells in vitro --- p.94 / Chapter 3.2.2 --- Indirubin-3’-oxime exhibited no significant cytotoxicity on normal cells --- p.101 / Chapter 3.2.3 --- Indirubin-3’-oxime induced G0/G1 cell cycle arrest in LA-N-1 cells --- p.103 / Chapter 3.2.4 --- Indirubin-3’-oxime caused mitochondrial dysfunction in LA-N-1 cells --- p.106 / Chapter 3.2.5 --- Indirubin-3’-oxime selectively reduced ERR γ and PGC-1β protein and mRNA levels in LA-N-1 cells --- p.111 / Chapter 3.2.6 --- Indirubin-3’-oxime induced cell cycle arrest at G0/G1 phase and apoptosis of SH-SY5Y cells --- p.113 / Chapter 3.2.7 --- Indirubin-3’-oxime reduced mitochondrial mass and elevated mitochondrial ROS level in SH-SY5Y cells --- p.115 / Chapter 3.2.8 --- Indirubin-3’-oxime increased the caspase 8, caspase 9 and caspase 3/7 activities in SH-SY5Y cells --- p.117 / Chapter 3.3 --- Discussion --- p.119 / Chapter Four / Modulatory effects and action mechanisms of tryptanthrin on human neuroblastoma cells --- p.125 / Chapter 4.1 --- Introduction --- p.126 / Chapter 4.2 --- Results --- p.129 / Chapter 4.2.1 --- Tryptanthrin inhibited the cell growth and colony formation of human neuroblastoma cells --- p.129 / Chapter 4.2.2 --- Tryptanthrin exhibited no significant cytotoxicity on normal cells --- p.136 / Chapter 4.2.3 --- Tryptanthrin induced cell cycle arrest at G0/G1 phase --- p.138 / Chapter 4.2.4 --- Tryptanthrin induced apoptosis of LA-N-1 cells --- p.140 / Chapter 4.2.5 --- Tryptanthrin induced morphological neuronal differentiation in LA-N-1 cells --- p.143 / Chapter 4.2.6 --- Tryptanthrin induced the expression of neuronal differentiation markers --- p.146 / Chapter 4.2.7 --- Tryptanthrin down-regulated the expression of N-myc in LA-N-1 cells --- p.149 / Chapter 4.3 --- Discussion --- p.152 / Chapter Five / Anti-angiogenesis effects and action mechanisms of indirubin-3'-oxime and tryptanthrin --- p.158 / Chapter 5.1 --- Introduction --- p.159 / Chapter 5.2 --- Results --- p.163 / Chapter 5.2.1 --- Indirubin-3’-oxime and tryptanthrin inhibited the proliferation of endothelial cells --- p.163 / Chapter 5.2.3 --- Indirubin-3’-oxime and tryptanthrin reduced the tube formation of endothelial cells --- p.168 / Chapter 5.2.4 --- Indirubin-3’-oxime and tryptanthrin blocked angiogenesis in the in vivo Matrigel plug model --- p.171 / Chapter 5.2.5 --- Indirubin-3’-oxime and tryptanthrin reduced the angiogenic gene expression in endothelial cells --- p.174 / Chapter 5.2.6 --- Indirubin-3’-oxime and tryptanthrin attenuated VEGFR2-mediated signaling pathways in endothelial cells --- p.176 / Chapter 5.2.7 --- Indirubin-3’-oxime bound to the ATP-binding site of VEGFR2 kinase domain --- p.181 / Chapter 5.2.8 --- Tryptanthrin bound to the ATP-binding site of VEGFR2 kinase domain --- p.182 / Chapter 5.3 --- Discussion --- p.184 / Chapter Six / Conclusions and future perspectives --- p.191 / References --- p.206

Submitted by Verena Bastos (verena@uefs.br) on 2015-08-05T22:08:06Z No. of bitstreams: 1 Disserta??o Mona Oliveira vers?o final Maio2003 PPGbiotec.pdf: 4793209 bytes, checksum: 5e62150796f4527c8d492f92de5041e2 (MD5) / Made available in DSpace on 2015-08-05T22:08:06Z (GMT). No. of bitstreams: 1 Disserta??o Mona Oliveira vers?o final Maio2003 PPGbiotec.pdf: 4793209 bytes, checksum: 5e62150796f4527c8d492f92de5041e2 (MD5) Previous issue date: 2013-05-08 / Funda??o de Amparo ? Pesquisa do Estado da Bahia - FAPEB / Glioblastomas (GBMs) are the most common and aggressive primary tumors of the CNS. The survival of patients with this diagnosis remains very low, with poor prognosis even after surgical therapy associated with radiotherapy and chemotherapy. The present work carried out a screening for 24 synthetic ?alkaloid-like? to determine their effects on cell viability of quimioresistentes human (Gl-15 and U251) glioblastoma cells and murine (C6) glioma cells. Among the alkaloids tested (100?M) RLB87 was the most cytotoxic for transformed cells, inhibiting the viability in 75.0% 97.2% 76.6% of GL-15, U251 and C6 cells, respectively, after 72 h exposure, and it did not show toxicity to normal glial cells. It was also observed that RLB87 promoted apoptosis, 24 and 72 h after treatment, in a time-dependent manner. Moreover RLB87 also inhibited cell migration and proliferation with cells arrest at G0/G1 phase, since 24 h after treatment. Additionally, the cytotoxicity of four RLB87 analogues was tested in view to elucidate important aspects in chemical structure, required for its activity. We observed positive correlation between cytotoxic effect and isomeric of phenyl functions, with ester function and also lipophilicity. These finding suggest the ?alkaloid-like? RLB87 as a promising anticancer agent as well as a prototype for new agents for treatment of malignant and recurrent gliomas. / Glioblastomas (GBMs) s?o os tumores prim?rios mais comuns e agressivos do SNC. A sobrevida dos pacientes com esse diagn?stico continua muito baixa, tendo progn?stico ruim mesmo ap?s terapia cir?rgica seguida de radio e quimioterapia. No presente trabalho, foi realizada a prospec??o de 24 mol?culas de s?ntese, alkaloids-like, para determina??o de seus efeitos sobre a viabilidade de c?lulas quimioresistentes de glioblastoma humano (GL-15 e U251) e murina (C6). Entre os compostos testados ? (100JM), RLB87 foi o mais citot?xico para c?lulas transformadas, inibindo a viabilidade em 75,0%, 97,2%, 76,6% da GL-15, U251 e C6, respectivamente e o mesmo n?o apresentou toxicidade para c?lulas gliais normais. Observou-se, que o RLB87 promoveu apoptose 24 e 72 h ap?s o tratamento de forma tempo-dependente. O RLB87 igualmente inibiu a prolifera??o celular com acumulo na fase G0/G1 do ciclo celular ap?s 24 h. A migra??o das c?lulas de glioma foi tamb?m inibida ap?s tratamento com RLB87. Adicionalmente 4 an?logos do RLB87 foram tamb?m avaliados, elucidando aspectos importantes na estrutura qu?mica, requeridos para sua atividade, que possuem correla??o positiva com regioisomeria das fenilas, presen?a da fun??o ?ster e lipofilicidade. O RLB87 ? apresentado como promissor agente antineopl?sico assim como um prot?tipo para novos agentes terap?uticos para o tratamento de gliomas malignos e recidivados.

Yip, Hon Yan Kelvin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 218-242). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vii / CHINESE ABSTRACT (摘要） --- p.xi / PUBLICATIONS --- p.xiv / TABLE OF CONTENTS --- p.xv / Chapter CHAPTER 1: --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Neuroblastoma --- p.2 / Chapter 1.1.1 --- An overview of neuroblastoma --- p.2 / Chapter 1.1.2 --- Epidemiology of neuroblastoma --- p.3 / Chapter 1.1.3 --- Clinical presentations of neuroblastoma --- p.5 / Chapter 1.1.4 --- Diagnosis and clinical assessment of neuroblastoma --- p.8 / Chapter 1.1.5 --- Staging of neuroblastoma --- p.10 / Chapter 1.1.6 --- Genetic aberrations of neuroblastoma --- p.12 / Chapter 1.1.7 --- Therapies of neuroblastoma --- p.15 / Chapter 1.2 --- Banlangen alkaloids --- p.20 / Chapter 1.2.1 --- An overview of Banlangen alkaloids --- p.20 / Chapter 1.2.2 --- "Pharmacokinetics of indirubin, tryptanthrin and their derivatives" --- p.24 / Chapter 1.2.2.1 --- Bioavailability of indirubin and its derivatives --- p.24 / Chapter 1.2.2.2 --- Toxicity of indirubin and its derivatives --- p.25 / Chapter 1.2.2.3 --- Bioavailability of tryptanthrin --- p.26 / Chapter 1.2.2.4 --- Toxicity of tryptanthrin --- p.27 / Chapter 1.2.3 --- "Pharmacological effects of indirubin, tryptanthrin and their derivatives" --- p.28 / Chapter 1.2.3.1 --- Selective inhibitor on kinases --- p.29 / Chapter 1.2.3.2 --- Anti-inflammatory activities --- p.31 / Chapter 1.2.3.3 --- Anti-tumour activities --- p.32 / Chapter 1.2.3.3.1 --- Anti-leukemic activity --- p.32 / Chapter 1.2.3.3.2 --- Apoptosis-inducing activity --- p.34 / Chapter 1.2.3.4 --- Anti-viral properties --- p.37 / Chapter 1.2.3.5 --- Anti-microbial properties --- p.37 / Chapter 1.3 --- Aims and Scope of This Study --- p.39 / Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.41 / Chapter 2.1 --- Materials --- p.42 / Chapter 2.1.1 --- Cell lines --- p.42 / Chapter 2.1.2 --- "Cell culture media, reagents and buffers" --- p.43 / Chapter 2.1.3 --- General staining solutions --- p.46 / Chapter 2.1.4 --- Drugs and chemicals --- p.47 / Chapter 2.1.5 --- Reagent for primary cultures preparation --- p.48 / Chapter 2.1.6 --- Reagents for cell proliferation assay --- p.48 / Chapter 2.1.7 --- Reagents for DNA extraction --- p.50 / Chapter 2.1.8 --- Reagents for gel electrophoresis of nucleic acids --- p.51 / Chapter 2.1.9 --- Reagents and buffers for flow cytometry --- p.53 / Chapter 2.1.10 --- Reagents and buffers for measuring caspase activity --- p.54 / Chapter 2.1.11 --- "Reagents, buffers and materials for Western blot analysis" --- p.58 / Chapter 2.1.12 --- Reagent for Hoechst staining --- p.68 / Chapter 2.2 --- Methods --- p.69 / Chapter 2.2.1 --- Culture of cell lines --- p.69 / Chapter 2.2.2 --- Determination of cell viability --- p.70 / Chapter 2.2.3 --- Determination of cell proliferation by tritiated thymidine ([ 3H]-TdR) incorporation assay --- p.72 / Chapter 2.2.4 --- "Isolation, culture and cytotoxicity test of murine peritoneal macrophages" --- p.73 / Chapter 2.2.5 --- "Isolation, culture and cytotoxicity test of murine bone marrow cells" --- p.74 / Chapter 2.2.6 --- Cytotoxicity test of primary cortical neurons from SD rats --- p.75 / Chapter 2.2.7 --- Determination of colony forming ability --- p.75 / Chapter 2.2.8 --- Analysis of cell cycle profile /DNA content by flow cytometry --- p.76 / Chapter 2.2.9 --- Detection of DNA fragmentation by agarose gel electrophoresis --- p.77 / Chapter 2.2.10 --- Quantitative detection of DNA fragmentation by Cell Death ELISAplus kit --- p.78 / Chapter 2.2.11 --- Detection of intracellular reactive oxygen species (ROS) generation --- p.80 / Chapter 2.2.12 --- Measurement of caspase activity --- p.81 / Chapter 2.2.13 --- Hoechst 33342 staining --- p.83 / Chapter 2.2.14 --- Cell morphological study --- p.83 / Chapter 2.2.15 --- Analysis of morphological changes by flow cytometry --- p.84 / Chapter 2.2.16 --- Assay for acetylcholine esterase (AChE) activity --- p.85 / Chapter 2.2.17 --- Protein expression study --- p.86 / Chapter 2.2.18 --- Statistical analysis --- p.89 / Chapter CHAPTER 3: --- IN VITRO STUDIES ON THE ANTI PROLIFERATIVE EFFECT OF INDIRUBIN-3'-OXIME AND TRYPTANTHRIN ON NEUROBLASTOMA CELLS --- p.90 / Chapter 3.1 --- Introduction --- p.91 / Chapter 3.2 --- Results --- p.95 / Chapter 3.2.1 --- Effects of indirubin-3'-oxime and tryptanthrin on the proliferation of human and the murine neuroblastoma cells --- p.95 / Chapter 3.2.2 --- Kinetic and reversibility studies of the anti-proliferative effect of indirubin-3'-oxime and tryptanthrin on the murine neuroblastoma Neuro-2a BU-1 cells --- p.107 / Chapter 3.2.3 --- Cytotoxic effect of indirubin-3'-oxime and tryptanthrin on the murine neuroblastoma Neuro-2a BU-1 cells --- p.115 / Chapter 3.2.4 --- Effects of indirubin-3'-oxime and tryptanthrin on the clonogenicity of the murine neuroblastoma Neuro-2a BU-1 cells --- p.120 / Chapter 3.2.5 --- Cytotoxicity of indirubin-3'-oxime and tryptanthrin on primary cells --- p.123 / Chapter 3.2.6 --- Effects of tryptanthrin on the cell cycle profile and expression of cyclins and cyclin-dependent kinases (CDKs) in the murine neuroblastoma Neuro-2a BU-1 cells --- p.132 / Chapter 3.2.7 --- Effect of indirubin-3'-oxime on the cell cycle profile in the murine neuroblastoma Neuro-2a BU-1 cells --- p.133 / Chapter 3.3 --- Discussion --- p.142 / Chapter CHAPTER 4: --- IN VITRO STUDIES ON THE APOPTOSIS-INDUCING EFFECT OF INDIRUBIN-3'-OXIME ON NEUROBLASTOMA CELLS --- p.150 / Chapter 4.1 --- Introduction --- p.151 / Chapter 4.2 --- Results --- p.156 / Chapter 4.2.1 --- Induction of DNA fragmentation in the indirubin-3'-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.156 / Chapter 4.2.2 --- Induction of chromatin condensation in the indirubin-3 '-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.160 / Chapter 4.2.3 --- Induction of caspase activities in the indirubin-3 '-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.162 / Chapter 4.2.4 --- Induction of Reactive Oxygen Species (ROS) in the indirubin-3' -oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.169 / Chapter 4.2.5 --- Expression of pro-apoptotic and anti-apoptotic proteins in the indirubin-3 '-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.173 / Chapter 4.3 --- Discussion --- p.177 / Chapter CHAPTER 5: --- STUDIES ON THE DIFFERENTIATION-INDUCING ACTIVITY OF TRYPTANTHRIN ON NEUROBLASTOMA CELLS --- p.188 / Chapter 5.1 --- Introduction --- p.189 / Chapter 5.2 --- Results --- p.193 / Chapter 5.2.1 --- Effects of tryptanthrin on the cell size and cellular complexity of the murine neuroblastoma Neuro-2a BU-1 cells --- p.193 / Chapter 5.2.2 --- Morphological studies on tryptanthrin-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.196 / Chapter 5.2.3 --- Effect of tryptanthrin on the acetylcholine esterase (AChE) activity in the murine neuroblastoma Neuro-2a BU-1 cells --- p.198 / Chapter 5.2.4 --- Effects of tryptanthrin on the expression of tau protein and other mediators involved in the differentiation pathway --- p.200 / Chapter 5.3 --- Discussion --- p.204 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.209 / REFERENCES --- p.218